Abstract
Introduction:
There are no published randomized data on secondary prevention in humans about whether aspirin affects nitric oxide (NO) formation. In patients with chronic stable coronary disease, we tested whether aspirin at clinically relevant doses increases NO formation.
Materials and Methods:
In a randomized, double-blind trial, 37 patients from 2 cardiology office practices were assigned to daily doses of 81, 162.5, 325, 650, or 1300 aspirin for 12 weeks. Primary prespecified outcome measures were changes in heme oxygenase (HO-1), a downstream target of NO formation, and asymmetrical dimethyl arginine (ADMA), a competitive inhibitor of NO synthase.
Results:
There were no significant differences for HO-1 or ADMA between any of the clinically relevant doses of aspirin tested, so all were combined. For HO-1, there was a significant increase (10.29 ± 2.44, P < .001) from baseline (15.37 ± 1.85) to week 12 (25.66 ± 1.57). The mean ratio (MR) of week 12 to baseline for HO-1 was significantly higher than 1.0 (1.67, confidence interval [CI] from 1.60 to 1.74, P < .001). For ADMA, there was a significant decrease (−0.24 ± 0.11, P < .001) from baseline (0.78 ± 0.08) to week 12 (0.54 ± 0.07). The MR of week 12 to baseline for ADMA was significantly lower than 1.0 (0.69, CI from 0.66 to 0.73, P < .001).
Conclusions:
In patients with chronic stable coronary disease, all clinically relevant daily doses of aspirin tested, from 81 to 1300 mg, produce similar and statistically significant increases in HO-1 and decreases in ADMA. These are the first randomized data on secondary prevention patients. These data support the hypothesis that aspirin has additional beneficial effects mediated through NO formation. Further research, including direct randomized comparisons on atherosclerosis using noninvasive techniques as well as on occlusive vascular disease events, is necessary.
Introduction
Although the antiplatelet effects are sufficient to explain the observed reductions by aspirin in risks of occlusive cardiovascular disease (CVD) events, 1,2 nitric oxide (NO) may play a prominent role in the inhibition of atherosclerosis, 3 and aspirin may affect NO. 4,5
Specifically, NO has a number of intracellular effects that lead to vasorelaxation, endothelial regeneration, inhibition of leukocyte chemotaxis, and platelet adhesion. Endothelium damage induced by atherosclerosis leads to the reduction in bioactivity of endothelial NO synthase (eNOS), with subsequent impaired release of NO together with a local enhanced degradation of NO by increased generation of reactive oxygen species with subsequent cascade of oxidation-sensitive mechanisms in the arterial wall. Further, many commonly used vasculoprotective agents have their therapeutic actions through the production of NO. 3 –5 Heme oxygenase (HO-1) is a cytoprotective downstream target and is considered a surrogate for biologically active NO. 6 Asymmetric dimethyl arginine (ADMA) is a competitive inhibitor of NOS. 7
Aspirin is capable of activating the NO-cyclic guanosine monophosphase (cGMP) signaling pathway in endothelial cells. Increases in NO and cGMP have been demonstrated to improve the integrity of the endothelium. 8 This novel pathway may have functional relevance and, if so, might significantly contribute to aspirin-induced prevention of endothelial injury in occlusive CVD. In the light of these observations, activation of endothelial NO formation could be an important mechanism by which aspirin, in addition to its antiplatelet effects, reduces the risks of occlusive vascular diseases. We have previously provided the first randomized evidence in high-risk primary prevention patients with metabolic syndrome that aspirin, at 5 clinically relevant doses tested from 81 mg to 1300 mg daily, increases nitric oxide (NO) formation. 4
We conducted the first randomized trial in secondary prevention patients designed a priori to test whether these 5 clinically relevant doses of aspirin increase NO as measured by increases of HO-1 or deceases in ADMA.
Materials and Methods
In a randomized, double-blind trial, 37 secondary prevention patients (25 men; aged 46 to 80 years inclusive) with chronic stable coronary disease taking daily 81 mg enteric-coated aspirin (ECA) were randomized to daily doses of 81 mg, 162.5 mg, 325 mg, 650 mg, or 1300 mg of plain aspirin for 12 weeks. In these patients who had been treated for at least 1 year previously with 81 mg ECA prescribed by their cardiologists, we had not prespecified that randomization to plain aspirin, at all these daily doses, would produce similar significant reductions in thromboxane B2 and prostaglandin E2 at 12 weeks. These post hoc data from a randomized trial contribute to the formulation of the hypothesis that patients who take low-dose ECA may not attain the clinical benefits of aspirin on occlusive CVD events. 9
We defined chronic stable coronary disease as patients without a prior event for over 1 year and who had 1 of the following 5 criteria: angiographic evidence of 70% or greater stenosis; previous percutaneous coronary intervention (PCI); coronary artery bypass graft (CABG); history of a myocardial infarction; or positive exercise test.
In addition, women of child-bearing potential had to be using a medically acceptable method of contraception (oral contraception, depo-provera injection, intrauterine device, condom with spermicide, diaphragm, cervical cap, progestin implant, abstinence, tubal ligation, oopherectomy, total abdominal hysterectomy) throughout the entire trial period.
We excluded the following patients: Patients taking more than 81 mg aspirin daily. Patients taking any of the following medications for less than 3 months or who plan to take them for the first time during the next 3 months: angiotensin converting enzyme (ACE) inhibitors, angiotensin receptor blockers, calcium channel blockers, or statins. Patients within 1 year of a coronary intervention, including PCI or CABG. Patients with a planned coronary intervention. Patients taking antiplatelet drugs such as clopidogrel or nonsteroidal anti-inflammatory drugs or anticoagulant drugs such as warfarin. Patients who are currently cigarette smokers. Patients who are pregnant, planning to become pregnant, nursing a child, or taking hormone replacement therapy. Patients with any coagulation, bleeding, or blood disorders. Patients who are sensitive or allergic to aspirin. Patients with documented history of any gastrointestinal disorders including bleeding ulcers. Patients with atrial fibrillation. Patients with any evidence of cancer or kidney, liver, lung, blood, or brain disorders. Patients with any abnormal laboratory value or physical finding that, in the view of the responsible clinician, may interfere with interpretation of the trial results, be indicative of an underlying disease state, or compromise the safety. Patients with class IV heart failure. Patients with severe aortic insufficiency or aortic regurgitation.
At baseline and 12 weeks, antecubital venous blood samples were obtained for HO-1 and ADMA. The HO-1 assays were performed using enzyme-linked immunosorbent assay (ELISA; Millipore Corporation, Billerica, Massachusetts; R & D Systems, Minneapolis, Minnesota; Sigma-Aldrich Corp, St Louis, Missouri). Each sample was measured in triplicate, and the overall intra-assay coefficient of variation was 2.1% ± 0.3%, with a plasma recovery rate between 85% and 99%.The ADMA assays were performed by ELISA (ALPCO Diagnostics, Salem, New Hampshire). Each sample was measured in triplicate, and the overall intra-assay coefficient of variation was 7.2% ± 1.2%, with a plasma recovery rate between 85% and 99%. Analysis of variance was used to test for the significance of differences between doses at baseline and at week 12 as well as for the paired differences between the 2 time points for HO-1 and ADMA. Due to missing data, 35 participants had both baseline and week 12 measures for both HO-1 and ADMA. Since there were no significant differences in dose, we pooled all dose levels and used a paired Student t test to determine whether there were significant differences between baseline and 12 weeks for HO-1 and ADMA. We also used paired Student t tests to determine whether there were significant modifications in the effects of aspirin by age (above or below the median), gender (men or women), and race (Caucasian, African American, or other). For HO-1 and ADMA, we calculated the ratios of the means, or mean ratios (MR), from week 12 to baseline. For each MR, we calculated 95% confidence intervals (CI) by computer simulation derived from the estimated distributions of each outcome. All significance tests were conducted using a 2-sided α level of .05.
Role of the Funding Source
This trial was funded as an investigator-initiated grant to Florida Atlantic University (FAU), with Charles H. Hennekens, MD, DrPH, and Sir Richard Doll, Research Professor, as the principal Investigators, by Bayer. The funding source, Bayer, had no role in the design, conduct, analysis, interpretation, preparation of the manuscript, or the decisions about whether or where to submit the manuscript for publication.
Results
Despite the relatively small sample size, randomization achieved a fairly balanced distribution of baseline characteristics by treatment group. Among the notable baseline characteristics were mean age of 64.0 (with a median of 63.7) years and mean body mass index (BMI) of 30.6. The vast majority of these patients with chronic stable coronary disease were being treated according to various guidelines with statins (86%), ACE inhibitors (54%), and β adrenergic blockers (76%; Table 1).
Baseline Characteristics by Randomized Daily Dose of Aspirin.a
Abbreviations: BMI, body mass index; PCI, percutaneous coronary intervention; ACE, angiotensin-converting enzyme; ANOVA, analysis of variance; CABG, coronary artery bypass graft; SD, standard deviation.
aReported as mean (SD) or frequency (%).
b P value from ANOVA test or Fisher exact test based on data type.
There were no significant differences between any of the 5 doses of aspirin tested for HO-1 and ADMA (Table 2). For HO-1, there was a significant increase (10.29 ± 2.44, P < .001) from baseline (15.37 ± 1.85) to week 12 (25.66 ± 1.57). Specifically, the MR of week 12 to baseline for HO-1 was significantly higher than 1.0 (1.67, CI from 1.60 to 1.74, P < .001; Table 3). There were no significant modifications in the effects of aspirin on HO-1 by age (P = .267) or gender (P = .416). For ADMA, there was a significant decrease (−0.24 ± 0.11, P < .001) from baseline (0.78 ± 0.08) to week 12 (0.54 ± 0.07). Specifically, the MR of week 12 to baseline for ADMA was significantly lower than 1.0 (0.69, CI from 0.66 to 0.73, P < .001; Table 3). There were no significant modifications in the effects of aspirin on ADMA by age (P = .287) but a possible, nonsignificant, greater decrease in ADMA over time for men (−0.27 ± 0.11) than in women (−0.18 ± 0.11; P = .062).
Lack of Statistically Significant Differences Between Each Clinically Relevant Dose of Aspirin on Markers of Nitric Oxide (NO) Formation.a
Abbreviations: ADMA, asymmetrical dimethyl arginine; HO-1, heme oxygensase.
aAll P values > .05.
bHO-1 was measured in nanograms per millilter.
cADMA was measured in micromoles per liter.
Statistically Significant Differences Between Baseline and 12 Weeks for all 5 Clinically Relevant Doses of Aspirin on Markers of Nitric Oxide (NO) Formation.
Abbreviations: ADMA, asymmetrical dimethyl arginine; HO-1, heme oxygensase; CI, confidence interval; SD, standard deviation.
a P value from paired t test of change in each variable from baseline to 12 weeks.
bHO-1 was measured in nanograms per millilter.
cADMA was measured in micromoles per liter.
Discussion
These are the first randomized data on secondary prevention in humans that aspirin increases NO formation. In addition, these effects are apparent across a wide range of usual doses of aspirin from 81 to 1300 mg daily used in clinical practice, including cardiovascular disease as well as pain relief. Since all randomized patients had chronic stable coronary disease, there was no placebo group; so it is possible, at least in theory, that these effects are simply a spontaneous change over time. The magnitude of the effects, however, makes this possibility extremely unlikely. The trial was designed a priori to test whether aspirin increases NO formation and, if so, whether there was effect modification by dose. In this randomized trial of patients with chronic stable coronary disease, aspirin, at all 5 clinically relevant doses tested, increases NO formation. Although there is no apparent dose effect, the sample sizes were small. Nonetheless, with respect to the primary prespecified hypothesis, aspirin produces significant increase in HO-1, a downstream target of NO formation, as well as a significant decrease in ADMA, a competitive inhibitor of NOS. The only previously published randomized data in humans were among high-risk primary prevention patients.
Although the results for the primary prevention patients and secondary prevention patients were virtually identical and statistically significant, a meta-analysis revealed some importantly relevant additional findings. Specifically, at baseline the primary prevention patients (29.37 ± 2.13) had significantly higher mean levels of HO-1 than that of the secondary prevention patients (15.37 ± 1.85; P < .001). At week 12, the mean HO-1 was also significantly higher in primary prevention patients (57.45 ± 5.6) than in the secondary prevention patients (25.66 ± 1.57; P < .001). For HO-1, there was neither a dose effect (P = .359) nor a dose by trial interaction effect (P = .329). At baseline, primary prevention patients (1.70 ± 0.22) also had significantly higher levels of ADMA than that of the secondary prevention patients (0.78 ± 0.08; P < .001). At week 12, the mean ADMA level was significantly higher in primary prevention patients (0.81 ± 0.10) than in the secondary prevention patients (0.54 ± 0.07; P < .001). For ADMA, there was neither a dose effect (P = .530) nor a dose by trial interaction effect (P = .820). In the primary prevention patients, the utilization rates of statins, other lipid-lowering agents, ACE inhibitors, diuretics, β-blockers, and calcium channel blockers were all relatively low. We, therefore, had speculated whether similar beneficial effects of aspirin on HO-1 and ADMA would be present in patients with higher utilization rates of these drugs. The present data indicate that aspirin increases NO formation in secondary prevention patients with high rates of utilization of these drugs.
Conclusions
These data on secondary prevention provide the first randomized evidence in humans that contribute importantly relevant and complementary information to the hypothesis that aspirin has additional beneficial effects mediated through NO formation. Such effects, if proven to be clinically relevant, would be adjuncts to the well-documented and well-accepted antiplatelet properties of aspirin 1,2 that are sufficient to explain the reductions in risks of occlusive CVD events. Although there is controversy about the role of coronary plaque burden and its location in particular segments of the coronary arteries as predictors of clinical CVD events, there is general consensus that plaque burden represents a good index of atherosclerosis and its progression. In numerous investigations, cardiac computerized tomographic angiography is safe and detects significant changes in plaque progression over 2 years. 10 The availability of such randomized data in humans might justify a large-scale randomized trial to provide the first direct test of this hypothesis on clinical CVD events. Thus, further research, including direct randomized comparisons on atherosclerosis and other clinical events due to occlusion, which may be performed using various noninvasive techniques, is warranted to test whether this hypothesis has clinical or public health relevance.
Footnotes
Authors’ Notes
The trial had an independent Data and SafetyMonitoring Board (DSMB), which consisted of Peter Libby, MD, Marc A. Pfeffer, MD (Chair), and Bernard Rosner, PhD. The trial also had an independent Statistical Data Analysis Center at the Department of Biostatistics and Informatics at the University of Wisconsin with David DeMets, PhD, as Principal Investigator and ScottHetzel, MS, as Project Director. The trial was registered at www.clinicaltrials.gov as “Aspirin Dose and Atherosclerosis in Patients with Heart Disease.” The registration number is NCT00272337.
Declaration of Conflicting Interests
The author(s) declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: Professor Hennekens reported that he was funded for 5 years by FAU as Principal Investigator on two investigator-initiated research grants funded to FAU by Bayer; that he is currently funded by the Charles E. Schmidt College of Medicine at FAU; that he serves as an independent scientist in an advisory role to investigators and sponsors as Chair of Member of Data and Safety Monitoring Boards for Actelion, Amgen, AstraZeneca, Bayer, Bristol-Myers Squibb, British Heart Foundation, Cadila, Canadian Institutes of Health Research, Lilly, Sunovion and the Wellcome Foundation; that he serves as an independent scientist in an advisory role to the United States (U.S.) Food and Drug Administration, U.S. National Institutes of Health, Children's Services Council of Palm Beach County and UpToDate; that he serves as an independent scientist in an advisory role to legal counsel for Stryker; that he receives royalties for authorship or editorship of three textbooks and as co-inventor on patents -concerning inflammatory markers and cardiovascular disease which are held by Brigham and Women's Hospital; that he has an investment management relationship with The West-Bacon Group within SunTrust Investment Services who has discretionary investment authority and that he does not own any common or preferred stock in any pharmaceutical or medical device company.
Funding
The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This trial was funded as an investigator-initiated grant to Florida Atlantic University (FAU), with Charles H. Hennekens, MD, DrPH Sir Richard Doll Professor, as Principal Investigators by Bayer. The funding source, Bayer, had no role in the design, conduct, analysis, interpretation, preparation of the manuscript, or any decisions concerning submission or publication.